Project Summary There is an urgent need to develop new therapeutic options for metastatic breast cancer. The goal of this proposal is to help fulfill this need by shedding new light on the molecular pathways driving breast cancer progression and metastasis. Our recently published data indicate that AKT1 phosphorylates S326 of heat shock factor 1 (HSF1), the transcription factor known as the master regulator of the heat shock response, leading to HSF1 activation. S326-activated HSF1 increases expression of Slug, an important mediator of epithelial-to-mesenchymal transition (EMT), which is an early step in metastasis. Our preliminary data further indicate that S326-phosphorylated HSF1 was frequently detected in breast cancer metastases. Pharmacologic inhibition of HSF1 synergizes with an AKT inhibitor to reduce breast cancer cell growth and cancer stem cell self-renewal. These results led us to hypothesize that S326-activated HSF1 contributes to breast cancer progression and the dual targeting of HSF1 and AKT inhibits breast tumor growth and progression. To fully address this hypothesis, we will complete the following three Specific Aims. (1) Determine the effects of HSF1 S326 phosphorylation on breast cancer progression. First, we will assess the effects of S326 phosphorylation on HSF1 activity in breast cancer and the role of different AKT isoforms in HSF1 activation. Second, we will utilize in vitro and in vivo models of breast cancer and in patient tumors to determine the impact of HSF1 S326 phosphorylation on breast tumor growth, progression, and metastasis. (2) Investigate whether pharmacologic inhibition of HSF1, alone and in combination with AKT inhibition, suppresses breast cancer growth and metastasis. First, we will determine whether combined inhibition of HSF1 and AKT targets the treatment-resistant tumor initiating cells using in vitro and in vivo models of breast cancer. Second, we will determine whether this novel treatment strategy suppresses primary breast tumor growth and metastasis using multiple animal models. (3) Identify an AKT-HSF1 pathway activation gene expression signature and use it to predict breast cancer clinical progression and patient outcomes. First, we will identify a gene expression signature indicative of AKT-HSF1 pathway activation. Second, we will use this signature to examine the association between HSF1 activation and patient outcomes. This proposal is significant because it will gain a deeper understanding of breast cancer progression, investigate a novel combination treatment for aggressive breast cancer, and develop a gene expression signature that informs clinical progression and outcomes. This project has conceptual, translational, and technical innovations as it will: i) examine the role of HSF1 as a novel mediator of metastasis, ii) investigate a new therapeutic regimen that simultaneously targets HSF1 and AKT, and iii) develop an HSF1 pathway activation gene expression signature as a novel prognostic indicator for breast cancer survival and response to chemotherapy.